1. Field
The present technology pertains to the field of “hot starts” for enzymes that act upon nucleic acids, such as for example, polymerases.
2. Description of the Related Art
The invention of the polymerase chain reaction (PCR) has made DNA diagnostics and forensics possible. Saiki et al., “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.” Science, 230, 1350-1354 (1985).(1) By adding the reverse transcriptase present in retroviruses to the reaction, the utility of PCR was expanded to RNA.
A common problem with PCR is non-specific amplification of nucleic acids during PCR set-up and before the initial denaturation and amplification steps. The non-specific activity of the enzymes, for example, at room temperature can result in unwanted background products and the formation of primer-dimers. These unwanted products can interfere with generating the desired amplicons and can provide false signals when using PCR as a diagnostic tool. Many enzymes that act upon DNA, including DNA polymerases and reverse transcriptases can suffer from problems with non-specific activity.
Low specificity during PCR set up for the polymerase has been overcome through the use of various “hot start” methods. Such methods include, the use of temperature controlled magnesium concentration, and the use of temperature activated dNTP's and primers. The most commonly available reagents commercially utilize chemically modified or antibody inhibited polymerases.
Reverse transcriptase (RT) is capable of adding bases to the 3′ ends of the primers during room temperature PCR setup even in the absence of RNA.(2) The extra bases added to the primers can inhibit amplification of the intended target, leading to false negatives and/or causing primer-dimers. While some of the above-described methods could conceivably be adapted for temperature dependent activation of the reverse transcriptase, most require temperatures that would denature the RT and consequently are not viable for use with RT.
While various PCR “hot start” technologies exist, embodiments described herein provide new approaches, including approaches that can be used with RT PCR, as well as other enzymes that act upon nucleic acid molecules.